A new endophyte Monascus ruber SRZ112 as an efficient production platform of natural pigments using agro-industrial wastes

A number of biopigment applications in various industrial sectors are gaining importance due to the growing consumer interest in their natural origin. Thus, this work was conducted to valorize endophytic fungi as an efficient production platform for natural pigments. A promising strain isolated from leaves of Origanum majorana was identified as Monascus ruber SRZ112 produced several types of pigments. The nature of the pigments, mainly rubropunctamine, monascin, ankaflavin, rubropunctatin, and monascorubrin in the fungal extract was studied by LC/ESI–MS/MS analyses. As a first step towards developing an efficient production of red pigments, the suitability of seven types of agro-industrial waste was evaluated. The highest yield of red pigments was obtained using potato peel moistened with mineral salt broth as a culture medium. To increase yield of red pigments, favourable culture conditions including incubation temperature, incubation period, pH of moistening agent, inoculum concentration, substrate weight and moisture level were evaluated. Additionally, yield of red pigments was intensified after the exposure of M. ruber SRZ112 spores to 1.00 KGy gamma rays. The final yield was improved by a 22.12-fold increase from 23.55 to 3351.87 AU g−1. The anticancer and antioxidant properties of the pigment’s extract from the fungal culture were also studied. The obtained data indicated activity of the extract against human breast cancer cell lines with no significant cytotoxicity against normal cell lines. The extract also showed a free radical scavenging potential. This is the first report, to our knowledge, on the isolation of the endophytic M. ruber SRZ112 strain with the successful production of natural pigments under solid-state fermentation using potato peel as a substrate.

Natural pigments consist of several types of bioactive compounds used extensively in several applications of biopigments in different industrial sectors, as food colorants, dietary supplements, pharmaceuticals, cosmetics, aquaculture and poultry feed 1 . Currently, natural pigments in comparison to the synthetic colors have taken a lead in a rapidly changing industry. The number of synthetic colorants allowed by the regulatory agencies is quite dependent of the compound and application purpose. So, their number has decreased due to their potential carcinogenicity as well as their undesirable mutagenicity. The European Union has recommended in 2010 the acceptable levels of some colorings for consumption every day. Also, the World Health Organization and US Food and Drug Administration limited the usage of synthetic colors in drugs and food products 2 . Moreover, a chemical synthesis of pigments is a complex process and generates unwanted by-products and hazardous wastes affecting the environment 3 . Nowadays, extraction of natural pigments from plants and animals have gained more attention 4 ; however, it is an expensive process with very low yield and environmentally costly 5 . By 2024, it was expected that the world color market used in food industry will reach approximately 5.7 billion US dollar 6 . Unfortunately, the basic science and industry still have not overcome the high cost and low availability of natural pigments 7 . Therefore, the demand for the naturally produced biopigments will continue to rise. Microbial pigments, among the natural pigments, showed several advantages including the ease of scaling up and harvest 8 as well as not subjected to the nature vagaries 9 . Fungal platforms for production of biopigments are of great interest as environmentally safer substitutes for synthetic ones. Fungi, in comparison with algal and bacterial platforms, are more efficient biotechnology agents due to the high mycelial growth rate 10  www.nature.com/scientificreports/ 20 days, initial pH of the MSB (adjusted using 1 N NaOH or 1 N HCl in the range 3.0-8.0), inoculum concentration (10 4 -10 8 spore/mL), substrate weight per 250-mL flask (5-30 g/flask), and moisture level (50-90%, w/w).
Effect of 60 Co gamma-irradiation on growth and red pigment production. Spore suspension of the Monascus ruber SRZ112 was prepared and irradiated by gamma rays in the range 0.25-4.00 KGy using a 60 Co Gamma chamber (Model: MC20, Russia). Immediately following irradiation, suspensions were stored in darkness (to avoid photoreactivation) at 4 °C overnight. After which, the irradiated suspensions were separately tested for red pigment production using 15 g potato peel moistened by MSB at 70% moisture level, incubated at 30 °C for 10 days, the most favorable conditions for red pigment production from the previous experiment.
Analytical methods. Estimation of the fungal growth. Growth of the Monascus ruber SRZ112 was estimated by quantifying the released N-acetyl glucosamine from the acid-hydrolyzed chitin in the fungal cell wall 26 . Glucosamine concentrations were expressed as mg g −1 dry fermented substrate and estimated after recording the UV absorption at 530 nm from a standard curve 16 .
Extraction of red pigment. Red pigment was extracted according to a procedure reported previously with slight modifications 27 . At the end of the incubation period, 10 mL of absolute ethanol was added to 1 g of fermented substrate. Then, the mixture was incubated for 4 h with an agitation speed of 200 rpm. After that, the mixture was filtered through Whatman paper No 1 and further centrifuged at 45×g for 10 min. The supernatant was diluted and used for estimation of the red pigments yield.
Determination of the red pigment yield. Red pigment production was evaluated by spectrophotometric measurements (JENWAY-305, UK) at 500 nm in terms of absorbance units per g dry fermented substrate (AU g −1 ) according to a previously reported method 28 , taking into consideration the dilution factor of the sample 29 . Red pigment yield was estimated and expressed as absorbance units which were multiplied by the dilution factor 30 .

Chemical identification of pigments by LC/ESI-MS/MS analysis.
The crude pigment extract of the gamma irradiated M. ruber SRZ112 was subjected to LC/ESI-MS/MS analysis to identify the present chemical constituents. LC/ESI-MS/MS measurements were performed by a RSLC Dionex UltiMate 3000 (Thermo Fisher Scientfic, USA) with mass spectrometer ESI-Q-TOF, maXis impact (Bruker, USA) with Syncronis Phenyl C18 1.7 µm, 100 × 2.1 mm, column (Thermo Scientific, USA). The mobile phase was a mixture of 0.5% aqueous formic acid v/v (A) and 0.5% formic acid in acetonitrile (B), the flow rate was 0.2 mL/min, column incubation temperature was 30 °C. The program was: 0 min-80% A, 20% B; 0-10 min-50% A, 50% B; 10.0-14. Determination of the anticancer activity. In order to evaluate the cytotoxicity of the crude pigment extract, the ethanol extracts were pooled, evaporated and dissolved in 0.1% DMSO to obtain a desired range of concentrations. Cytotoxicity of the pigments extracted from SSF culture of M. ruber SRZ112 was evaluated against normal human melanocytes (HFB-4) and human breast carcinoma (MCF-7) by the MTT-based assay 31 . In brief, both normal human melanocytes and human breast carcinoma cells were treated separately with 20 μL of the prepared crude pigment extract. After-which, the MTT solution was added to the wells, and the absorbance was recorded at 570 nm using a microplate reader (SunRise, TECAN, Inc., USA). Wells containing cells treated with 0.1% DMSO (without pigments) served as controls. The relative viability of the treated cells was estimated by the following equation: The IC 50 values, 50% inhibitory concentration, were calculated using GraphPad Prism software, San Diego, CA.
Determination of the antioxidant activity. In order to evaluate the antioxidant potential of the crude pigment extract, the ethanol extracts were pooled, evaporated and dissolved in methanol to obtain a desired range of concentrations. Antioxidant potential of the crude pigments extracted from SSF culture of M ruber SRZ112 was estimated by the free radical scavenging assay 32 using 2,2-diphenyl picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis, MO, USA). Simultaneously, a positive control of ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) was also tested at the same concentrations. Scavenging activity (%) was calculated as the change in the absorbance of the mixture (DPPH + pigments) with respect to the DPPH solution only (control).

Statistical analysis. Calculated mean is for triplicate measurements from two independent experiments.
Statistical significance of the obtained means was analyzed by the analysis of variance (One-Way ANOVA) and the Least Significant Difference (LSD) tests (at 0.05 level) using SPSS software version 22 (IBM Corp). www.nature.com/scientificreports/

Results
Isolation and identification of the fungal strain. The pigment producing fungal strain namely SRZ112 was isolated from healthy leaves of the medicinal herb Origanum majorana. Figure 1 presented morphological features with growing colonies of the SRZ112 strain on Czapek's-yeast autolysate agar plates. After 10 days of growth at 25 °C, red pigmentation was observed in both front (Fig. 1A) and reverse ( Fig. 1B) view of the growing colonies. In addition, chains of a sexual conidia, young ascoma (Fig. 1C), and the mature ascoma with ellipsoidal ascospores (Fig. 1D) were observed under microscope. Identification of the SRZ112 strain was confirmed by amplification and sequencing of 18S rRNA of the SRZ112 strain. The retrieved sequences were deposited (under accession number MT140350.1) in the GenBank. Using BLAST tools, the retrieved sequences and those of the closely related species accessed from the GenBank were compared. Figure 2 presented the constructed phylogenetic tree indicating that the SRZ112 strain had a 100% identity with Monascus ruber strains. Table 1 showed that growth of M. ruber SRZ112 and red pigments yield were greatly influenced by the used waste and the type of moistening agent. Among the tested wastes, potato peel moistened by MSB was the best solid substrate. The recorded values of growth and red pigment concentration were 10.21 ± 0.32 mg g −1 and 23.55 ± 0.83 AU g −1 , representing the highest yield of red pigments. Moreover, rice bran followed by sugarcane bagasse were also good substrates for red pigment production by M. ruber SRZ112. On using rice bran moistened by MSB, values of the growth and red pigment were 8.66 ± 0.22 mg g −1 and 20.64 ± 0.42 AU g −1 . Also, these values were 7.32 ± 0.39 mg g −1 and 17.78 ± 0.21 AU g −1 , on using sugarcane bagasse. However, rice husk, corn cobs, and wheat husk were very poor substrate for either the growth of M. ruber SRZ112 or production of red pigment (Table 1).  www.nature.com/scientificreports/ Effect of different cultivation conditions on production of red pigment. Generally, the experimental results indicated that both of the growth and red pigments production by M. ruber SRZ112 were greatly affected by changing the cultivation conditions (Figs. 3, 4, and 5). It was noticed that there was a significant increase (P ≤ 0.05) in growth of M. ruber SRZ112 and production of red pigment at all the tested conditions. Among the tested incubation temperatures 30 °C was the most proper for maximum yield of red pigment where a significant yield enhancement (P ≤ 0.05) was obtained. Conversely, fermentation that was conducted either at the higher temperature extreme (40 °C) or at lower ones (20 °C) reduced the obtained red pigment yield from M. ruber SRZ112 (Fig. 3A). Optimum incubation period for maximum red pigment production was recorded after 10 days (Fig. 3B) at using moistening agent with pH value adjusted at 6.0 (Fig. 4A). The most favorable inoculum concentration for red pigments yield was observed at 10 7 spore/mL (Fig. 4B). Changing substrate weight (potato peel) dramatically affected the red pigment production specially at a 15 g where maximum yield was attained (Fig. 5A). The highest yield of red pigments was achieved on using potato peel moistened by MSB at 70%, w/w moisture level (Fig. 5B).

Suitability of agro-industrial wastes for maximum red pigment production. Data presented in
Production enhancement of red pigment using 60 Co gamma irradiation. Exposure of the M.
ruber SRZ112 spores to several doses of gamma rays was used to study their effects on the production of red pigments. Results illustrated in Table 2 clearly confirmed the gamma rays' dose-related manner effect on the growth of M. ruber SRZ112. It was also noticed from Table 2 that the highest reduction in the growth was recorded after receiving higher doses at 2.00 KGy. In addition, the M. ruber SRZ112 spores failed to grow after exposure to 4.00 KGy. However, cultures from spores receiving 1.00 KGy promoted the highest red pigment yield. Significant differences (P ≤ 0.05) in the recorded values of the red pigment yield were found at this dose and the yield reached  Chemical identification of pigments produced by M. ruber SRZ112. Based on the LC/ESI-MS/ MS spectra (Fig. 6), five different compounds were determined as well as their names and structures were also assigned. From the obtained data, the recorded [M+H] + values were 359.1867 for monascin (Fig. 6A), 355.1537 for rubropunctatin (Fig. 6B), 387.2158 for ankaflavin (Fig. 6C), 383.1847 for monascorubrin (Fig. 6D), and 354.1703 for rubropunctamine (Fig. 6E). Moreover, no fragments corresponding to the mycotoxin citrinin were detected.
Anticancer and antioxidant properties of pigments extracted from M. ruber SRZ112 culture. Table 3 showed that pigments extracted from cultures of the endophyte M. ruber SRZ112 grown on potato peel were active against the malignant MCF-7 breast cancer cell lines meanwhile no significant cytotoxic activity towards normal human melanocytes (HFB-4). The least inhibitory concentration of pigments extract against MCF-7 cell lines was 10 µg mL −1 . Data also showed that the increase in pigment concentration resulted in a significant decrease in MCF-7 cell proliferation indicating the concentration-dependent cell death. Data in Table 3 also showed that the estimated IC 50 value of the pigment extract against the MCF-7 cell lines was 64.32 µg mL −1 .  and red pigments production (AU g −1 ) by Monascus ruber SRZ112 under SSF. Initial pH of the MSB moistening agent was adjusted to varying values using 1 N NaOH and HCl. Solidstate grown cultures were carried out on potato peel at 30 °C for 10 days. All data are shown as the mean ± SD of triplicate measurements from two independent experiments. www.nature.com/scientificreports/ Testing the antioxidant potential of pigments extracted from cultures of the endophyte M. ruber SRZ112 grown on potato peel clearly confirmed their antioxidant activity (Table 3) when compared by ascorbic acid at all the tested different concentrations. The recorded data confirmed the concentration-dependent manner of pigments extract in inhibition of free radicals. Data also revealed that the least inhibitory concentration of both ascorbic acid and pigment extract was 10 µg mL −1 . Table 3 showed that the estimated IC 50 values of pigment extract and ascorbic acid were 211.51 and 72.08 µg mL −1 , respectively.

Discussion
Nature provides several sources of pigments, despite that the high cost of their extraction and processing limits their availability, problems that industry still has not overcome. Thus, there are pressing scientific and social needs to promote the use of natural pigments as a safer and greener alternative for synthetic colors to reduce their health risks. Moreover, biopigments has challenged all the biotechnologists to search, characterize, develop a cost-effective and eco-friendly strategies for their production 19 . In this study, an endophytic fungus SRZ112 was isolated and found to produce pigments. The studied morphological characteristics of the SRZ112 were identical with universal keys 23,24 , concerning the identification of Monascus ruber. Also, molecular studies of the sequence from the SRZ112 strain revealed a 100% identity with those of M. ruber. In the literature, the are no data on the isolation of any endophytic Monascus ruber strains. As such, information and experience on using endophytic fungi as sources for production of pigments are very scarce 33,34 . Nevertheless, our study is the first report, to our knowledge, on the isolation of M. ruber from plant tissues. Generally, over 20 species of Monascus were reported; however, only few M. ruber strains have been recognized [35][36][37] .
Microbial pigments not only add color, but they also have interesting biological properties such as antioxidant 38 , antimicrobial 39 , anticancer, anti-inflammatory 19 , antiproliferative, and immunosuppressive activities 40 . Because of these pharmacological properties, this study was extended to evaluate the cytotoxic and antioxidant properties of pigments extracted from the culture of M. ruber SRZ112. Our results confirmed the antioxidant effect of pigments extracted from the fungal cultures. Moreover, our results indicated that pigments Figure 5. Effect of different weights of potato peel as substrate (A) and effect of different moisture levels (B) on growth (mg g −1 ) and red pigments production (AU g −1 ) by Monascus ruber SRZ112 under SSF. Initial pH of the MSB moistening agent was adjusted to 6.0 using 1 N NaOH and HCl. Solid-state grown cultures were carried out on potato peel at 30 °C for 10 days. All data are shown as the mean ± SD of triplicate measurements from two independent experiments. Table 2. Survival rate (%), fungal growth (mg glucosamine g −1 dry fermented substrate) and red pigment production (AU g −1 dry fermented substrate) of M. ruber SRZ112 grown under SSF at different doses of gamma irradiation. Solid-state grown cultures were grown on potato peel moistened with MSB (pH 6.0) incubated at 30 °C for 10 days. Calculated mean is for triplicate measurements from two independent experiments ± SD, a-f means with different superscripts in the same column either for carbon or nitrogen sources are considered statistically different (LSD test, P ≤ 0.05).

Gamma-irradiation dose (Gy)
Survival rate (%) Glucosamine concentration (mg g −1 ) Red pigment yield (AU g −1 ) 0 www.nature.com/scientificreports/ extracted from cultures of the endophyte M. ruber grown on potato peel were active against the malignant MCF-7 breast cancer cell lines meanwhile no significant cytotoxic activity towards normal human melanocytes (HFB-4). The same selective behavior was previously reported against normal diploid fibroblast cells 41 and normal gastric epithelial cells 42 . Meanwhile, both of the pigment-rich fractions and the whole extract of the commercial Chinese red fermented rice showed inhibitory activities against colon cancer 43,44 . Based on the promising anticancer and antioxidant properties of the pigments extract of M. ruber SRZ112, chemical constituents were investigated by LC/ESI-MS/MS analysis. Our results confirmed the presence of the following pigments; rubropunctatin (orange) monascin (yellow), ankaflavin (yellow), monascorubrin (orange), and rubropunctamine (red). The obtained data were in agreement with previous reports concerning the chemical characterization of the five pigments 21,44,45 . Moreover, no fragments corresponding to the mycotoxin citrinin were detected confirming that the produced culture extract is citrinin-free. Among these pigments, the red is of specific interest due to its potential for several therapeutic applications including antioxidant, immunosuppressive,  Table 3. Cytotoxic and antioxidant activities of the crude pigment extracted from SSF cultures of M. ruber SRZ112 grown on potato peel. Calculated mean is for triplicate measurements from two independent experiments ± SD, a-d Means with different superscripts in the same column are considered statistically different (LSD test, P ≤ 0.05).

(C)
100.00 ± 0.00 a 100.00 ± 0.00 a 0.00 ± 0.00 d 0.00 ± 0.00 d 5 100.00 ± 0.00 a 100.00 ± 0.00 a 0.00 ± 0.00 d 0.00 ± 0.00 www.nature.com/scientificreports/ antimicrobial 21 , anti-tumor, and cytotoxic potentials 45 . As such, red is the most common color used in food industry 19 . Thus, this study was directed to improve the red pigment yield by the M. ruber SRZ112 strain. As a first step, several agro-industrial residues were evaluated as cultural substrates for the production of red pigment by the endophytic fungus M. ruber SRZ112 grown under solid state fermentation. Over the years, SSF proved to be the most proper environment for a fungal growth 46 and production of elevated rates of several metabolites of industrial significance application 47 . In particular, the possibility of using wastes generated by food and industry 48 as well as the lower energy demand turned SSF into an economically viable and eco-friendly technology 49,50 .
In this study, our results revealed that a noticeable variation in the obtained yield of red pigment was recorded according to the tested waste and moistening agent. Potato peel moistened by the MSB promoted the highest yield by M. ruber SRZ112. In the literature, a wide range of substrates were used for production of red pigments by several Monascus strains 19 . However, our study is the first report, to our knowledge, on using potato peel as a culture substrate for production of red pigments by M. ruber SRZ112. According to Liang and McDonald, the main components of potato peel waste (on dry basis) were 30% non-starch polysaccharide, 25% starch, 18% protein, 20% acid-soluble and acid-insoluble lignin, 1% lipids, and 6% ash 51 . Generally, the specific nature of the cultural substrate and composition of the moistening agent during a SSF had great influences on the process of red pigment production, as supported by several reports ( 44 , and references therein).
Results of testing the effect of different cultivation conditions on the production of red pigment by M. ruber SRZ112 confirmed that the SSF process was greatly affected by changing the cultivation conditions as there is a significant increase in growth and red pigments yield. Of all the tested conditions, fermentation conducted at 30 °C for 10 days using moistening agent with pH value adjusted at 6.0 inoculated with inoculum concentration of 10 7 spore/mL with 15 g potato peel moistened by MSB at 70%, w/w moisture level promoted the highest yields of red pigments. Monascus sp. is of a mesophilic nature which need moderate incubation temperature for optimal growth and maximum production rates 52 . Moreover, temperature between 30 and 37 °C was the most proper range for several Monascus isolates 27 . Conversely, high temperatures over 35 °C in rare cases were found the optimum for higher rates of pigment production 53 . In accordance with our results, Pawanjot stated that 10 days of incubation was the optimum for the production of Monascus pigments 54 . The author further reported a reduction in the pigment yield due to contamination. Similarly, the yield of pigment was increased with the incubation period; however, the prolonged incubation resulted in contaminations, depletion of medium ingredients, so the yield was reduced 55 . In partial agreement with our results, the favorable pH for red pigment production was at 5.5-8.5 56 . Moreover, higher pH (over 6.0) promoted synthesis of red pigment whereas lower pH (below 3.5) promoted synthesis of yellow pigments 45 . In agreement with our results, the highest pigment yield was achieved using an inoculum concentration between 10 6 and 10 7 spores/mL 54 . Moreover, the pigment productivity was decreased at elevated inoculum concentration due to the production of excess biomass 27 and depletion of nutrients essential for pigment synthesis 55 . In agreement with our results, previous reports confirmed that moisture level and substrate weight greatly control the aeration of the SSF process 27,55 . Low-level moisture level adversely affects the metabolic activity by reducing nutrients solubility. In addition, the lower moisture can cause low degree of swelling of the substrate particles increasing the water tension and transfer of oxygen to the system 48,49 .
Herein, red pigment yield from M. ruber SRZ112 was significantly intensified after exposure to 1.00 KGy gamma rays. The obtained red pigment yield at this dose was 22.12 times higher than that obtained from the non-irradiated cultures. Exposure to mutagenic agents is a traditional method for stain improvement 21 . It mainly uses chemical, physical, and biological mutagens to induce mutations in the microbial genes, which can artificially guide biosynthesis of metabolic pathways and accumulate the desired metabolic product. For example, a mutant strain of M. purpureus designed as M630 with high pigments production was induced by exposure to room temperature plasma 57 . Generally, exposure to physical mutagens such as gamma irradiation 58 and UV light 59,60 may have a stimulatory effect on production of red pigment M. ruber SRZ112. Results obtained in this study also confirmed the negative effect of gamma-irradiation on the growth of M. ruber SRZ112. In accordance with these results, previous studies reported the same observation on the reducing effect of gamma radiation on the fungal growth 61,62 . In the literature, gamma rays as a mutagenic agent was highly recommended several reports 61,62 for improvement of several fungal strains over-expressing a wide array of metabolites of industrial significance 63,64 . It is worthy here to mention that, several Monascus strains were reported in the literature with divers' productivities. For instance, Babitha and co-workers achieved 12.113 OD Units/gds by M. purpureus LPB 97 27 . Zhang and co-workers reported the yield of 6.63 U/g by M. purpureus AS3.531 65 . Carvalho and co-workers used Monascus for pigment production 216 AU/g dry substrates 53 . Sharmila and co-workers achieved a pigment yield of 7.18 ODU/mL 66 . Embaby and co-workers obtained a maximal value of 108.02 ODU/ml by M. purpureus ATCC16436 30 . Pawanjot produced natural red pigment using M. purpureus a color value of 85 ODU/g dm 54 .
In summary, a new Monascus strain with pigment producing ability was isolated from plant tissues and identified to our knowledge for the first time. The fungal extract showed promising anticancer and antioxidant potentials. Chemical characterizations of pigments produced by the fungal strain were investigated. For an efficient red pigment solid-state fermentation, several wastes were screened where potato peel promoted the highest red pigment productivity. The favorable cultural conditions for maximum production of red pigments were studied. Furthermore, red pigment yield by M. ruber SRZ112 was intensified to a 22.12-fold increase from the initial titers after exposure to gamma rays.

Data availability
All data generated or analyzed during this study are included in this published article.